Food Sci. Technol. Res., 6 (2), 106-1 14, 2OOO Antioxidative Constituents from Tessaria integnfolia Masateru ON0,l Chikako MASUOKA,2 Yusuke ODAKE,2 Satoshi IKEGASHIRA,2 Yasuyuki IToland Toshihiro NOHARA3 IResearch Institute of General Education and 2School ofAgriculture, Kyushu Tokai University, Choyo 5435, Aso, Kumamoto 869-1404, Japan jFaculty of Pharmaceutical Sciences, Kumamoto University, Oe-honmachi 5-1, Kumamoto 862-0973, Japan Received September 29, 1 999; Accepted February 1 1 , 2000 The methanol extract of the aerial part of Tessaria integnfolia (common name: pajaro bobo), which is used as an herb for the treatment of asthnra and liver injury in Peru, showed a stronger antioxidative activity than the standard synthetic antioxidant, 3-tert-butyl-4-hydroxyanisole (BHA). From this extract, five new eudesmane-type sesquiterpe- noids were isolated along with eighteen known compounds, and their structures were elucidated on the basis of MS, IH-NMR and 13C-NMR data. Among them, eleven phenolic compounds, four flavonoids, two lignans and five caffeoyl quinic acid derivatives were examined for their antioxidative activity using the ferric thiocyanate method, and nine compounds indicated a stronger antioxidative activity than the standard natural antioxidant, a-tocopherol at a con- centration of 0.5 mM. Especially, two flavonoids were identified as stronger antioxidants than BHA. The scavenging effect of the phenolic compounds on the stable radical 1,1-diphenyl-2-picrylhydrazyl was examined. All tested com- pounds showed this scavenging effect, with the effect of five caffeoyl quinic acid derivatives and three flavonoids being almost twice or more that of a-tocopherol at a concentration of 0.01 mM. Keywords : Tessaria integnfolia, Compositae, sesquiterpenoid, caffeoyl quinic acid derivative, flavonoid, Iignan, antioxidative effect Antioxidants are major constituents in protecting against lipid a ~ (ppm) scale with tetramethylsilane (TMS) as an internal stan- peroxidation which causes the rancidity of fats and oils in food. dard. The abbreviations used are as follows: s, singlet; br s, broad Antioxidants can also protect the human body from hannful free singlet; d, doublet; dd, double-doublet; br d, broad doublet; dq, radicals which damage unsaturated fatty acids in cell mem- double-quartet; qq, quarte-quartet; ddd, double-double-doublet; branes. DNA and enzymes. This damage is believed to induce ddq, double-double-quartet; dddd, double-double-double-dou- aging and various human diseases such as cancer, infiammation blet. Electron impact mass spectrum (EI-MS), negative ion fast and atherosclerosis (Yagi, 1987; Yoshikawa et al., 1 994). There- atom bombardment mass spectrum (negative FAB-MS) and pos- fore, the natural antioxidants from the herbs and edible plants, itive FAB-MS were obtained on a JEOL JMS-DX-303HF instru- which would be safer for human consumption than synthetic ment (JEOL). Optical rotations were measured with a JASCO antioxidants, may reduce the incidence of the human diseases in DTP- 1000 KUY digital polarimeter (JASCO, Tokyo). Visible association with free radicals. absorptions were measured with a Shimadzu UV- 1 40-02 spec- Tessaria integnfolia (Compositae) is used as an herb tea for trometer (Shimadzu, Kyoto). Column chromatography (CC) was the treatment of asthma and liver injury in Peru (Feo, 1 992; Shio- carried out over silica gel 60 (Merck, Art. 7734 and Art. 9385; ta, 1997). The presence of ~-amyrin acetate, P-selinene, squa- Merck, Darmstadt, Germany), Diaion HP 20 (Mitsubishi Chemi- lene, bisthienyl derivatives, Iignan, sesquiterpenoids, fiavonoids cal Industries Co.. Ltd.. Tokyo), MCI gel CHP 20P (Mitsubishi and caffeoyl quinic acid derivatives in this plant have been re- Chemical Industries Co., Ltd.), Sephadex LH20 (Pharmacia Fine ported (Bohlmann et al., 1977; Jakupovic et al. , 1985; Feo et al. , Chemicals, Uppsala, Sweden), Cosmosil 75C18 OPN (Nacalai 1990; Guerreiro et al. , 1990; Peluso et al., 1995). Tesque Inc., Kyoto) and Bio-Beads S-X2 (BIO-RAD Laborato- In the course of our studies on natural antioxidants (Ono et al. , ries, Hercules, CA). High perfoamance liquid chromatography 1995, 1997, 1998, 1999; Masuoka et al., 1997), the methanol (HPLC) was run on a micro pump LC- I OAS (Shimadzu) with an extract of the aerial part of this plant showed a stronger antioxi- RI Detector RID-6A (Shimadzu) or an UV-VIS detector SPD- dative activity than 3-tert-butyl-4-hydroxyanisole (BHA) using 1 OA (Shimadzu). For HPLC column chromatography, YMC the ferric thiocyanate method. Therefore, we reexamined the packed column AQ-324 S-5 120A ODS (YMC Co., Kyoto, 10 constituents from this extract. mm i.d. X250 mm; column 1), YMC Pack ODS-AQ ODS (YMC Co., 20 mm i.d. X250 mrn; column 2), Inertsil PREP- Materials and Methods ODS (GL Sciences, Tokyo, 20 mm i.d. X250 mm; column 3), The melting point was determined on a Yanagimoto micro- Wakosil-II 5C18 HG (Wako Pure Chemicals Indusuies, Ltd., melting-point apparatus and is uncorrected. Proton (1H)- and car- Osaka, 4.6 mm i,d. X250 mm; column 4), Kusano C.1.G. pre- bon- 1 3 ( 1 3C)-nuclear magnetic resonance (NMR) spectra were packed Si-5 (Kusano Kagakukikai Co., Tokyo, 22 mm i.d. X 100 recorded using a JEOL JNM-GX-400 and a JEOL alpha 500 mm; column 5) and Inertsil ODS-2 (GL Sciences, 6 mm i.d. spectrometer (JEOL, Tokyo), and chemical shifts were given on X250 mm; column 6) were used. High perfoamance thin layer chromatography (HPTLC) was performed on precoated silica gel E-mail mono @ as- I .ktokai-u,ac,jp plates (Merck, Art. 5628) using CHC13-MeOH-H20 (8 : 2 : 0.2) Antioxidative Constituents from Tessaria integnjb!ia 1 07 as developing solvent and detection was achieved by UV irradia- Fraction 41 was subjected to HPLC (columu I , 75~;~o MeOH; col- tion (254 nm, Atto Co., Ltd., Tokyo, SJ-103 IA) and by spraying umn 4, 65~;~. MeOH, in turn) to afford 15 (3 mg) and 18 (7 1 mg). the plates with 10% H2S04-MeOH reagent, followed by heating. HPLC (column 2, 60% MeOH) of fraction 42 furnished 19 (8 oL-Tocopherol, BHA, L-cysteine monohydrochloride monohy- mg). Fraction 34 was successively chromatographed over Bio- drate and I , I -diphenyl-2-picrylhydrazyl (DPPH) were obtained Beads S-X2 (hexane : AcOEt, I : I ) and HPLC (column 2, 60% from Nacalai Tesque Inc. Linoleic acid was purchased from MeOH) to give 20 (7 mg), 21 (18 mg) and 18 (32 mg). Fraction Tokyo Kasei Kogyo Co, (Tokyo). Tessaria integnfolia was pur- 35 was subjected to HPLC (column 2, 76~;~o MeOH; column I , chased from Fundation pala I a Investigacion Tecnologica del 70% MeOH, in turn) to furnish 17 (59 mg). HPLC (column 2, Recurso Agrobiologico Andio in Peru. 55~;~o MeOH) of fraction 37 gave 16 (1 1 mg), 12 (40 mg) and 22 Extraction and isolation The cut dried aerial part of Tes- (77 mg). Fraction 28 was subjected to MCI gel CHP 20P (50% saria integnfolia (2.00 kg) was extracted with MeOH (9.5 L, 8.3 MeOH-90% MeOH, gradient) CC to give I (62 mg) . Chroma- L) under refiux, and the solvent was removed under reduced tography of fraction 30 over MCI gel CHP 20P (H.O-MeOH, pressure to give a brown powder (175.5 g). This extract was sub- gradient) afforded 2 (40 mg). jected to Diaion HP 20 (60% MeOH, 80% MeOH. MeOH, ace- Chrysosplenol-D (1): yellow powder. EI-MS m/z: 360 [M]+. tone) CC to afford fractions I (98.19 g), 2 (26.58 g), 3 (30.70 g) IH-NMR (in DMSO-d6' 500 MHz) ~: 12.67 (1H, br s, OH-5), and 4 ( 1 7.98 g). Fraction I was partitioned between 1-BuOH 9.83 (1H, br s, OH), 9.40 (1H, br s, OH), 7.62 (lH, d, J=2.2 Hz, (200 ml, 100 ml, 100 ml) and H20 (300 ml). The combined 1- H-2'), 7.50 (1H, dd, J=2.2, 8.8 Hz. H-6'), 6.93 (1H, d, J=8.8 BuOH soluble fraction (53.87 g) was chromatographed over Hz, H-5'), 6.85 (1H, s, H-8), 3.93 (3H, s, OCH3), 3.81 (3H, s, Diaion HP 20 (H20, 10% MeOH, 40% MeOH, MeOH) to afford OCH3), 3.74 (3H, s, OCH3). 13C-NMR (in DMSO-d6' 100 MHz) fractions 5 (1 1.2 g), 6 (3.8 g), 7 (21.0 g) and 8 (16.8 g). Fraction ~: 178.1 (C-4), 158.5 (C-~), 155.9 (C-9), 151.6 (C-2 and C-5), 7 was subjected to silica gel (CHC13 : MeOH : H20, 14 : 2 : 0.1, 148.8 (C-4'), 145.2 (C-3'), 137.6 (C-3), 131.5 (C-6), 120.7 (C- 10 : 2 : 0.1, 8 : 2 : 0.2, 7 : 3 : 0.5, 6 : 4 : 1) CC to give fractions 9 1 '), 120.6 (C-6'), 1 15.6 (C-2' or C-5'), 1 15.5 (C-5' or C-2'), 105.5 (37 mg), 10 (214 mg), Il (l020 mg), 12 (217 mg), 13 (1426 (C-10), 91.2 (C-8), 60.0 (OCH3), 59.6 (OCH3), 56.4 (OCH3). mg), 14 (1800 mg) and 15 (1404 mg). Fraction 10 was subjected Quercetin (2): yellow powder. EI-MS m/z: 302 [M]+. IH- to HPLC (column 2, 50% MeOH; column 5, hexane-AcOEt, NMR (in DMSO-d6' 500 MHz) ~: 12.49 (lH, s. OH-5), 10.77 1 : I , in turn) to afford 23 (5 mg). HPLC (column 2, 40% (1H, s, OH), 9.56 (1H, br s, OH), 9.31 (1H, br s, OH), 9.28 (lH, MeOH) of fraction 1 2 gave 5 (26 mg). Fraction 14 was chro- br s.